1. Field Of The Invention
The present invention relates generally to the detection of protein and, more particularly, to a novel composition and method for the determination of protein in a biological sample utilizing phenolsulfonephthalein protein error indicator, buffer and an aliphatic polyether-polycarbonate present in an amount equal to or less than ten percent by weight.
2. Description Of The Background Art
The determination of the presence of protein in a biological sample is of the utmost importance in the diagnosis of several pathological conditions affecting the kidney, circulatory system and central nervous system. Accordingly, it is frequently necessary to quantitatively and/or qualitatively measure protein (albumin) in urine. This is especially important in the diagnosis of diabetes and kidney disease. The predominant protein in diabetes determinations is albumin; hence the model system for protein urine testing is albumin.
Methods for determining the presence of albumin in urine are well known. The most inexpensive and convenient method for albumin determination involves wetting a paper test strip impregnated with a protein error indicator with a small quantity of urine. If albumin is present in the urine sample, the test strip will indicate this by simply changing color. The color observed normally varies depending on the concentration of albumin in the sample. This variable color change is used to quantify the albumin in the sample.
While test strips of the above type are convenient and rapid vehicles for on-the-spot determinations of protein, one of the problems which has occurred is the so-called specific gravity effect. Typically tests for urinary protein use a pH indicator dye which shows a shift in pKa when complexed with protein (the "protein error" of pH indicators). This method does not provide sufficient separation between negative urine and those containing trace concentrations of protein (approximately 15 micrograms per deciliter) and gives false positive readings in urine of high specific gravity. It has been found that the addition of certain polycarbonate compounds to the protein error indicator both lowers the initial reactivity of the reagent paper and decreases the tendency for high specificity gravity urine samples to give a positive reading for protein.
Another problem which has occurred with the construction of such devices is that the absorbent material, usually filter paper, does not have consistent (i.e., uniform) reactivity with the reagents used in the system. Filter paper lots often interact with the reagents required for the determination of protein to a greater extent than the same carrier matrix material reacts with reagent compositions for the determination of other analytes.
The problems of adjusting the formulations to obtain uniform reactivity between the reagent composition and the carrier matrix material employed has been a long-standing problem in the field and one of significant importance to the industry. Initially it was believed that adjustment of the pH of the reagent composition would overcome the problem of the variability in the reactivity of the reagent composition and the carrier matrix material. pH adjustments, however, were not effective for all lots of carrier matrix material. The desired result was to achieve a reagent composition which would result in a lighter negative color and a darker positive color. Triton X-100, a polyethylene surfactant, was found to have an impact on reactivity but produced a lighter color at both the negative and positive ends of the color range.
Other means of adjusting reactivity were investigated. For example, all components of the reagent composition were varied but these variations were not found effective in producing the desired lighter negative color and a darker positive color. Polyvinyl alcohol also was introduced into the reagent composition and found to be ineffective in controlling reagent reactivity characteristics with differing lots of carrier matrix.
Thus, even with appropriate quality control and by trying to implement effective screening and invention/restrictions reactivity problems between differing lots of carrier matrix material could not be controlled effectively.
To overcome the reactivity problem mentioned above for the determination of protein an unique reagent composition was prepared and formulated in such a way that the variability in reactivity between the reagent composition and the carrier matrix has been substantially eliminated. Moreover, the reduction of urine pH variability on assay results is another observed benefit of the reagent composition.